Extension of the glycoprotein processing capabilities of the Lepidopteran insect cell line Spodoptera frugiperda by metabolic engineering.

摘要

Glycosylation, the addition of carbohydrates to a peptide backbone, is the most extensive co- and posttranslational modification of eukaryotic cells. The glycosylation profile of a recombinant glycoprotein can significantly affect its biological activity. This is particularly important when being used in human therapeutic applications. The baculovirus expression vector system (BEVS), used in conjunction with insect cell hosts, is a popular tool for the expression of heterologous proteins with an excellent track record of producing high levels of biologically active eukaryotic proteins. Insect cells are also capable of glycosylation, but it is generally believed that their N-glycosylation pathway is truncated in comparison with the pathway of mammalian cells. Studies of foreign glycoproteins expressed in insect cells have shown that the N-glycans are oligomannosylated and that insect cells are not capable of synthesizing fully elaborated complex or hybrid N-linked glycans. These results suggest that the requisite processing activities are either absent or present at levels too low to be generally effective.;In this study capillary electrophoresis (CE) was applied in combination with exoglycosidase digestion to analyze the structure of glycans derived from the total protein fraction collected from uninfected Spodoptera frugiperda Sf-9 insect cells. The glycans were analyzed by CE after exoglycosidase digestion through coinjection studies using fluorescently labeled standards. This analysis showed that the glycans were of the oligomannosylated type comigrating between hexasaccharide and undecasaccharide standards.;In the subsequent experiments, the N-glycosylation pathway was metabolically engineered by the addition of $b$1,4-galactosyltransferase under the control of an immediate early promoter (ie1 ), thereby extending the glycoprotein processing capabilities of insect cells. The analysis of the total protein fraction of AcMNPV-GalT infected Sf-9 cells, showed that the expression of recombinant $b$-1,4 galactosyltransferase enabled Sf-9 cells to synthesize galactosylated glycans.;Furthermore, the capability of the BEVS to express biologically active C1INH, a therapeutic glycoprotein, was demonstrated in a 10 L bioreactor. Sf-9 cells were infected with a recombinant baculovirus encoding the full C1INH gene under control of the polyhedrin promoter, resulting in a high yield of biologically active recombinant C1INH.